1. Field of the Invention
The present invention relates to transcription factor decoys for preventing cell apoptosis induced by endotoxin and use thereof, particularly to a DNA decoy for reducing cell apoptosis of endothelial cells induced by endotoxin. The present invention also relates to a pharmaceutical composition for curing or preventing cells from cell apoptosis induced by endotoxin, which comprises transcription factor decoy.
2. Description of the Prior Arts
Although progress and response of sepsis is becoming more predictable, there is still a need for effective therapy for sepsis (S. J. Streat, et al., World J. Surg., 2000, 24: 655-663). The mortality of sepsis patients is up to 26% in intensive care unit (ICU) and even up to 34% in high risk groups (C. Granja, et al., Crit. Care., 2004, 8: R91-R98). The symptom of sepsis includes shock, thrill, rapid breathing and heart rate and the like. Multiple organ failure is the major cause of high mortality, as high as 98%, in ICU (W. A. Knaus et al., Ann. Surg., 1985, 202: 685-693). Since sepsis ranks as the twelfth leading cause of death (D. L. Hoyert, et al., Natl. Vital Stat. Rep., 2001, 49: 1-113), development and improvement of therapy for sepsis is an urgent and important need.
Sepsis is a systemic inflammatory response (SIR) against bacterial infection (S. M. Dauphinee and A. Karsan, Lab Invest., 2006, 86: 9-22). The underlying mechanism of sepsis resides in serial responses of host and immune system caused by entrance of pathogen into blood, resulting in hyperdynamic state with increased cardiac output in an early phase, hypodynamic state with decreased cardiac output in a late phase, and finally a reduction of systemic vascular resistance and dilation of peripheral vessels, leading to hypotension and organ ischemia, so called septic shock (S. Yang, et al., Am. J. Physiol., 1999, 277: H1036-H1044). Since vasopressor is not effective against septic shock, sustained septic shock causes multiple organ dysfunction (MOD) and finally leads to multiple organ failure (MOF) and death (R. C. Bone, et al., The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine, Chest., 1992, 101: 1644-1655).
Recently, several studies have indicated that multiple organ failure induced by sepsis is caused by cell apoptosis (E. D. Papathanassoglou, et al., Crit. Care Med., 2000, 28: 537-549). Cell apoptosis is confirmed to be a significant mechanism against pathogens (H. Steller, Science., 1995, 267: 1445-1449). Sepsis is strongly associated with exogenous pathogen, bacterial endotoxin (S. M. Dauphinee and A. Karsan, Lab Invest., 2006, 86: 9-22).
Bacterial endotoxin is a major component of cell wall of Gram-negative bacteria, composed of polysaccharide and lipid. Therefore, endotoxin is also called lipopolysaccharide (LPS) (R. Pfeiffer, Untersuchungen uber das Choleragift, Medical Microbiology and Immunology, 1892, 11: 393-412). Pathophysiological mechanism of LPS-induced endotoxsemia resides in LPS entering a human body and engaging with CD14 on cell membranes of immune cells to form a complex and stimulating macrophages, monocytes and T lymphocytes of host immune system through downstream signaling to produce cytokines and inflammatory precursors such as prostaglandin (S. M. Dauphinee and A. Karsan, supra.; J. T. Rosenbaum, et al., Nature, 1980, 286: 611-613), resulting in inflammation, hyperplasia and apoptosis and finally sepsis. Current clinical therapies for sepsis includes hemodynamic monitoring, fluid replacement, pathogen control, antibiotic therapy, airway management, monitoring of renal function, blood sugar control and prevention of complication (A. P. Wheeler and G. R. Bernard, N. Engl. J. Med., 1999, 340: 207-214). All the above-mentioned monitoring and therapies are preventive and passive. No therapy exists that is immediately effective for severe sepsis or septic shock (S. J. Streat, et al., 2000, supra).
Only one drug for treating severe sepsis has been approved by the US Food and Drug Administration (FDA), activated protein C (APC). Clinical tests in 1960 sepsis patients with different sources of pathogens, ages, conditions and severity of organ dysfunction showed that activated protein C decreases 28-day mortality from 30.8% to 24.7%. (G. R. Bernard, et al., N. Engl. J, Med., 2001, 344: 699-709). In another clinical test, 70% of sepsis patients who were over 60 years old were selected and comprised 386 people, 40% of the 70% sepsis patients, who were over 75 years old were subjected to APC treatment and the 28-day and 2-year mortality was monitored. APC treatment was found to have no side effects in patients at different ages (C. J. Hinds, B. M. J., 2001, 323: 881-882). Therefore, the FDA approved APC for treating sepsis in Nov. 21, 2001.
Activated protein C is physiologically obtained by activating protein C (PC). PC is a vitamin K-dependent protein C, which is synthesized in the liver and contains a light chain of 155 amino acids and a heavy chain of 304 amino acids linked by disulfide bonds. PC belongs to serine protease family PC is activated by binding to thrombin (T) and thrombomodulin (TM) known as T-TM mechanism, which is regulated by endothelial protein C receptor (EPCR). APC exerts anticoagulant activity through cutting and inhibiting coagulation cofactors F VIIIa and coagulation cofactors F Va.
Receptor of APC on endothelial cells has been identified as proteinase-activated receptor 1 (PAR1). When PC is activated to APC, PAR1 and endothelial protein C receptor will both be expressed after PAR1 is activated by APC. Serial anti-inflammatory reactions are aroused by PAR1, which include suppression of p53, modulation of Bax/Bcl-2 and decrease of Caspase 3 signaling. These result in inhibiting IL-1, IL-6 and TNF-α synthesis to avoid cell apoptosis.
PAR is G protein-coupled receptor, including 7 transmembrane domains, and belongs to a family consisting of PAR1, PAR2, PAR 3 and PAR4, wherein PAR1, PAR3 and PAR4 can be activated by thrombin; and PAR2 can only be activated by trypsin, tryptase, and coagulation cofactor VIIa and Xa. The difference between PAR1 and other G protein is that activation of PAR1 is irreversible. Therefore, activated PAR1 enters lysosomes instead of being recycled to membrane. Due to PAR1's unique characters, PAR1 plays an important role in APC pathway.
Although APC has been confirmed as having a therapeutic effect on sepsis, use of APC on treatment of sepsis still has disadvantages. For example, percentage of severe hemorrhage occurring in sepsis patients increases from 2% to 3.5% and half-life of APC in blood is only 1.6 hour (G. R. Bernard, et al., N. Engl. J, Med., 2001, 344: 699-709). Collectively, in the field of the art there are no therapeutically effective pharmaceutical compositions or methods for treating sepsis. Accordingly, there is an urgent need for an effective and immediate novel therapy for sepsis.
To overcome the shortcomings, the present invention provides a composition and method for curing or preventing endotoxin-treated endothelial cells from apoptosis and also for treating sepsis to mitigate or obviate the aforementioned problems.